Glucan-based vaccines

ABSTRACT

Anti-glucan antibodies have been found to be protective against systemic fungal infection with  C. albicans , but the protective efficacy can be inhibited by blocking antibodies. The invention provides an immunogenic composition comprising a glucan and a pharmaceutically acceptable carrier, characterised in that, when administered to a mammalian recipient, the composition elicits protective anti-glucan antibodies but does not elicit antibodies which inhibit the protective efficacy of the anti-glucan antibodies. The glucan may be presented on the surface of a protease-treated microbial cell or may be presented as a protein-glucan conjugate. The glucan may be substituted by a glucan mimotope, a peptidomimetic of a glucan mimotope, or nucleic acid encoding a mimotope. Anti-glucan-antibodies show broad spectrum microbicidal activity. β-glucans are preferred, particularly those containing one or more β-1,6 linkages.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/701,250, filed Feb. 1, 2007, which is a divisional of U.S.application Ser. No. 10/514,483, filed May 26, 2005, which is a 371filing of PCT/IB03/02460, filed May 15, 2003, which claims priority toGB 0211118.5, filed May 15, 2002, from which applications priority isclaimed pursuant to the provisions of 35 U.S.C. §§119/120, and whichapplications are hereby incorporated by reference in their entireties.

All documents cited herein are incorporated by reference in theirentirety.

TECHNICAL FIELD

The invention relates to vaccines, more particularly those againstfungal infections and disease.

BACKGROUND ART

Fungal infections are prevalent in several clinical settings,particularly in immunocompromised patients. The emergence of resistanceto antimycotics, in particular to the azoles, has increased interest intherapeutic and prophylactic vaccination against these fungi [1]. Amongfungal pathogens, Candida albicans is one of the most prevalent. Thisorganism is one of the principal agents of widespread opportunisticinfections in humans and causes candidiasis, a condition which is foundin both normal and immunocompromised patients. There have been severalattempts to provide anti-Candida vaccines.

There is widespread consensus in the field of medical mycology thatcellular immunity is critical for successful host defence against fungi[2], although the potential efficacy of humoral immunity in protectingagainst two major fungal pathogens (C. albicans and C. neoforzans) hasattracted attention [2,3]. For C. neoformans, antibodies to the capsularglucuronoxylomannan have been shown to mediate protection in animalmodels of infection. For C. albicans, cell-surface mannoproteins are thedominant antigenic components [1] of C. albicars and antibodies tomannan, proteases and a heat shock proteins have been associated withprotection against infection. Other vaccine candidates include: membersof the asparlyl proteinase (Sap2) family; the 65 kDa mannoprotein (MP65)[4]; adhesion molecules isolated from phosphomannan cell wall complexes[5]; peptides which mimic epitopes from the mannan portion of thephosphomannan complex of Candida [6]; and hemolysin-like proteins [7].

It is an object of the invention to provide further and better antigensfor inducing protective and/or therapeutic immune responses againstinfections, particularly against fungal infections.

DISCLOSURE OF THE INVENTION

Candida cells contain all non-secreted candidate protective antigensbut, even though they elicit high-level humoral and cell-mediated immuneresponses against various antigens, whole cell vaccines are ineffective.It has surprisingly been found that this low protective efficacy is notdue to the absence of immune responses to particular antigens, butrather to the presence of blocking antibodies in animal serum which caninteract with the intact fungus cell surface. In the absence of suchblocking antibodies, anti-glucan antibodies have been found to beprotective against systemic fungal infection, but the protective effectis inhibited when blocking antibodies are present. Fungal glucans arenaturally poor immunogens and have not previously been considered foreliciting protection.

Thus the invention provides an immunogenic composition comprising aglucan and a pharmaceutically acceptable carrier wherein, when it isadministered to a mammal, the composition elicits protective anti-glucanantibodies but does not elicit antibodies which inhibit the protectiveefficacy of the anti-glucan antibodies.

The Glucan

Glucans are glucose-containing polysaccharides found inter alia infungal cell walls. α-glucans include one or more α-linkages betweenglucose subunits and β-glucans include one or more β-linkages betweenglucose subunits.

α-glucans are found in various organisms, including S. mutans, which hasa cell wall containing both α-1,3- and α-1,6-glucans.

β-1,6-glucans occur frequently in fungi but are rarer outside fungi [8].Within a typical fungal cell wall, β-1,3-glucan microfibrils areinterwoven and crosslinked with chitin microfibrils to form the innerskeletal layer, whereas the outer layer consists of β-1,6-glucan andmannoproteins, linked to the inner layer via chitin and β-1,3-glucan. InC. albicans, 50-70% of the cell wall is composed of β-1,3- andβ-1,6-glucans. C. albicans does not contain β-1-2-glucan(s) orβ-1,4-glucan(s). Full length native β-glucans are insoluble and aregenerally branched.

The glucan used in accordance with the invention may comprise α and/or βlinkages. Where a linkages are present, the ratio of β linkages: αlinkages in the glucan will typically be at least 2:1 (e.g. 3:1, 4:1,5:1, 10:1, 20:1 or higher). In preferred embodiments, however, theglucan contains only β linkages.

β-glucans are preferred. The β-glucan may comprise one or moreβ-1,3-linkages and/or one or more β-1,6-linkages. It may also compriseone or more β-1,2-linkages and/or β-1,4-linkages. Particularly preferredare glucans containing one or more β-1,6-linkages.

The glucan may be branched.

Preferred glucans are β-glucans derived from the cell wall of a Candida,such as C. albicans. Other organisms from which β-glucans may be usedinclude Coccidioides immitis, Trichophyton verrucosum, Blastomycesdermatidis, Cryptococcus neoformans, Histoplasma capsulatum,Saccharomyces cerevisiae, Paracoccidioides brasiliensis, and Pythiumninsidiosum.

Preferred glucans are fungal glucans i.e. glucans found in fungi. A‘fungal’ glucan will generally be obtained from a fungus but, where aparticular glucan structure is found in both fungi and non-fungi (e.g.in bacteria, lower plants or algae) then the non-fungal organism may beused as an alternative source.

Full-length native β-glucans are insoluble and have a molecular weightin the megadalton range. It is preferred to use soluble glucans inimmunogenic compositions of the invention. Solubilisation may beachieved by fragmenting long insoluble glucans. This may be achieved byhydrolysis or, more conveniently, by digestion with a glucanase (e.g.with a β-1,3-glucanase or a β-1,6-glucanase). As an alternative, shortglucans can be prepared synthetically by joining monosaccharide buildingblocks.

Low molecular weight glucans are preferred, particularly those with amolecular weight of less than 100 kDa (e.g. less than 80, 70, 60, 50,40, 30, 25, 20, or 15 kDa). It is also possible to use oligosaccharidese.g. containing 60 or fewer (e.g. 59, 58, 57, 56, 55, 54, 53, 52, 51,50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40 39, 38, 37, 36, 35, 34, 33,32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4) glucose monosaccharide units.Within this range, oligosaccharides with between 10 and 50 or between 20and 40 monosaccharide units are preferred.

There are various sources of fungal β-glucans. For instance, pureβ-glucans are commercially available e.g. pustulan (Calbiochem) is aβ-1,6-glucan purified from Umbilicaria papullosa. β-glucans can bepurified from fungal cell walls in various ways. Reference 9, forinstance, discloses a two-step procedure for preparing a water-solubleβ-glucan extract from Candida, free from cell-wall mannan, involvingNaClO oxidation and DMSO extraction. The resulting product (‘Candidasoluble β-D-glucan’ or ‘CSBG’) is mainly composed of a linearβ-1,3-glucan with a linear β-1,6-glucan moiety. Further methods forpurifying β-glucans are disclosed in the examples herein, and ‘glucanghosts’ comprise high-purity β-glucans. β-1,3-glucans are known for useas health supplements [10].

As disclosed in the examples, preferred glucans are those obtainablefrom C. albicans, especially (a) β-1,6-glucans with β-1,3-glucan lateralchains and an average degree of polymerisation of about 30, and (b)β-1,3-glucans with β-1,6-glucan lateral chains and an average degree ofpolymerisation of about 4.

Pure β-glucans are, however, poor immunogens. For protective efficacy,therefore, β-glucans should be presented to the immune system inimmunogenic form. This may be achieved in various ways. In two preferredembodiments of the invention, the β-glucan included in the compositionof the invention is either: (a) a protease-treated and/ormannoprotein-depleted fungal cell which displays surface β-glucans; or(b) a glucan-carrier conjugate.

Protease-Treated Fungal Cells

β-glucans may be presented to the immune system on the surface of afungal cell. As β-glucans are not normally exposed in sufficientlyimmunogenic form on the surface of fungal cells, however, the cellsshould be treated with protease (e.g. a non-specific protease, such asProteinase K). Exposing fungi to protease in this way can depletemannoprotein and remove molecules which elicit blocking antibodies.

Thus the invention provides a protease-treated fungal cell havingsurface-exposed β-glucans. Preferably, the fungal cell's cell wall isfree or substantially free of mannoprotein.

The invention also provides an immunogenic composition comprising afungal β-glucan and a pharmaceutically acceptable carrier, wherein thefungal β-glucan is a component of a protease-treated fungal cell.Preferably, the fungal cell's cell wall is free or substantially free ofmannoprotein. More preferably, the composition as a whole is free orsubstantially free of mannoprotein.

The fungal cell is preferably a Candida and more preferably C. albicans.

Glucan-Carrier Conjugates

Glucans may be presented to the immune system in the form ofglucan-carrier conjugates. The use of conjugation to carrier proteins inorder to enhance the immunogenicity of carbohydrate antigens is wellknown [e.g. reviewed in refs. 11 to 19 etc.] and is used in particularfor paediatric vaccines [20].

The invention provides a conjugate of (i) a carrier protein and (ii) aglucan. The glucan is preferably a β-glucan as defined above, and ismore preferably a fungal β-glucan e.g. containing β-1,6 linkages.

The carrier protein may be covalently conjugated to the glucan directly,or a linker may be used.

Direct linkages to the protein may comprise oxidation of the glucanfollowed by reductive amination with the protein, as described in, forexample, references 21 and 22.

Linkages via a linker group may be made using any known procedure, forexample, the procedures described in references 23 and 24. A preferredtype of linkage is an adipic acid linker, which may be formed bycoupling a free —NH₂ group on an aminated glucan with adipic acid(using, for example, diimide activation), and then coupling a protein tothe resulting saccharide-adipic acid intermediate [15, 25, 26]. Anotherpreferred type of linkage is a carbonyl linker, which may be formed byreaction of a free hydroxyl group of a modified glucan with CDI [27, 28]followed by reaction with a protein to form a carbamate linkage. Otherlinkers include B-propionamido [29], nitrophenyl-ethylamine [30],haloacyl halides [31], glycosidic linkages [32], 6-aminocaproic acid[33], ADH [34], C₄ to C₁₂ moieties [35], etc.

Preferred carrier proteins are bacterial toxins or toxoids, such asdiphtheria or tetanus toxoids. These are commonly used in conjugatevaccines. The CRM₁₉₇ diphtheria toxoid is particularly preferred [36].Other suitable carrier proteins include the N. meningitidis outermembrane protein [37], synthetic peptides [38,39], heat shock proteins[40,41], pertussis proteins [42,43], protein D from H. influenzae [44],cytokines [45], lymphokines [45], hormones [45], growth factors [45],toxin A or B from C. difficile [46], iron-uptake proteins [47], etc. Itis possible to use mixtures of carrier proteins.

A single carrier protein may carry multiple different glucans [48].

When the conjugate forms the glucan component in an immunogeniccomposition of the invention, the composition may also comprise freecarrier protein [49].

After conjugation, free and conjugated glucans can be separated. Thereare many suitable methods e.g. hydrophobic chromatography, tangentialultrafiltration, diafiltration, etc. [see also refs. 50, 51 etc.].Tangential flow ultrafiltration is preferred.

The glucan moiety in the conjugate preferably an low molecular weightglucan or an oligosaccharide, as defined above. Oligosaccharides willtypically be sized prior to conjugation.

The protein-glucan conjugate is preferably soluble in water and/or in aphysiological buffer.

Antibodies

The invention provides a composition comprising (1) antibody whichrecognises a glucan and (2) a pharmaceutically acceptable carrier. Theglucan is preferably a β-glucan as defined above, and is more preferablya fungal β-glucan e.g. containing β-1,6 linkages.

The antibody is preferably a protective antibody, offering protectionagainst microbial infection and/or disease. The microbe may be a fungusor a bacterium, examples of which are given below.

The composition is preferably free or substantially free from antibodieswhich inhibit the protective efficacy of the anti-glucan antibodies. Forexample, where the glucan is a fungal β-1,6-glucan then the compositionis preferably free or substantially free from antibodies againstnon-glucan cell wall components, such as anti-mannoprotein antibodies.

The term ‘antibody’ includes any of the various natural and artificialantibodies and antibody-derived proteins which are available. Thus theterm ‘antibody’ includes polyclonal antibodies, monoclonal antibodies,Fab fragments, F(ab′)₂ fragments, Fv fragments, single-chain Fv (scFv)antibodies, oligobodies, etc.

Antibody-containing compositions of the invention may be used forpassive immunisation.

To increase compatibility with the human immune system, it is preferredto use human antibodies. As an alternative, antibodies of the inventionmay be chimeric or humanized versions of non-human antibodies [e.g.refs. 52 & 53].

In chimeric antibodies, non-human constant regions are substituted byhuman constant regions but variable regions remain non-human.

Humanized antibodies may be achieved by a variety of methods including,for example: (1) grafting complementarity determining regions (CDRs)from the non-human variable region onto a human framework(“CDR-grafting”), with the optional additional transfer of one or moreframework residues from the non-human antibody (“humanizing”); (2)transplanting entire non-human variable domains, but “cloaking” themwith a human-like surface by replacement of surface residues(“veneering”). In the present invention, humanized antibodies includethose obtained by CDR-grafting, humanizing, and veneering or variableregions. [e.g. refs. 54 to 60].

The constant regions of humanized antibodies are derived from humanimmunoglobulins. The heavy chain constant region can be selected fromany of the five isotypes: α, δ, ε, γ or μ.

Humanized or fully-human antibodies can also be produced usingtransgenic animals that are engineered to contain human immunoglobulinloci. For example, ref. 61 discloses transgenic animals having a humanIg locus wherein the animals do not produce functional endogenousimmunoglobulins due to the inactivation of endogenous heavy and lightchain loci. Ref. 62 also discloses transgenic non-primate mammalianhosts capable of mounting an immune response to an immunogen, whereinthe antibodies have primate constant and/or variable regions, andwherein the endogenous immunoglobulin-encoding loci are substituted orinactivated. Ref. 63 discloses the use of the Cre/Lox system to modifythe immunoglobulin locus in a mammal, such as to replace all or aportion of the constant or variable region to form a modified antibodymolecule. Ref. 64 discloses non-human mammalian hosts having inactivatedendogenous Ig loci and functional human Ig loci. Ref. 65 disclosesmethods of making transgenic mice in which the mice lack endogenousheavy chains, and express an exogenous immunoglobulin locus comprisingone or more xenogeneic constant regions.

Antibodies of the invention may be produced by any suitable means (e.g.by recombinant expression).

Mimotopes

Antigenic carbohydrates can be mimicked by polypeptides (‘mimotopes’)[e.g. 6, 66, 67, 68]. The invention also provides a polypeptidecomprising a mimotope of a glucan. The glucan is preferably a β-glucanas defined above, and is more preferably a fungal β-glucan e.g.containing β-1,6 linkages.

The mimotope preferably consists of at least 3 amino acids (e.g. atleast 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30 or more amino acids).

The polypeptide preferably consists of at least 3 amino acids (e.g. atleast 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 125, 150, 175, or at least200 amino acids).

The polypeptide preferably consists of no more than 250 amino acids(e.g. no more than 225, 200, 190, 180, 170, 160, 150, 140, 130, 120,110, 100, 95, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or even 5 amino acids).

Polypeptides consisting of between 6 and 20 amino acids are preferred.

Mimotopes of a glucan of interest may be identified in various ways. Apreferred technique for identifying a mimotope involves display (e.g.phage display) of a library of polypeptide sequences followed byselection of polypeptides which bind to antibodies specific for theglucan of interest. The selection procedure may be iterative in order tofocus on the best mimotopes.

Polypeptides of the invention may be prepared by various means.

A preferred method for production involves in vitro chemical synthesis[69, 70]. Solid-phase peptide synthesis is particularly preferred, suchas methods based on t-Boc or Fmoc [71] chemistry. Enzymatic synthesis[72] may also be used in part or in full.

As an alternative to chemical synthesis, biological synthesis may beused e.g. the polypeptides may be produced by translation. This may becarried out in vitro or in vivo. Biological methods are in generalrestricted to the production of polypeptides based on L-amino acids, butmanipulation of translation machinery (e.g. of aminoacyl-tRNA molecules)can be used to allow the introduction of D-amino acids or of othernon-natural amino acids, such as iodo-Tyr or methyl-Phe, azidohomo-Ala,etc. [73].

To facilitate biological peptide synthesis, the invention providesnucleic acid that encodes a polypeptide of the invention. The nucleicacid may be DNA or RNA (or hybrids thereof), or their analogues, such asthose containing modified backbones (e.g. phosphorothioates) or peptidenucleic acids (PNA). It may be single-stranded (e.g. mRNA) ordouble-stranded, and the invention includes both individual strands of adouble-stranded nucleic acid (e.g. for antisense, priming or probingpurposes). It may be linear or circular. It may be labelled. It may beattached to a solid support.

Nucleic acid according to the invention can, of course, be prepared inmany ways e.g. by chemical synthesis (e.g. phosphoramidite synthesis ofDNA) in whole or in part, by nuclease digestion of longer molecules,from genomic or cDNA libraries, by the use of polymerases etc.

The invention provides vectors (e.g. plasmids) comprising nucleic acidof the invention (e.g. expression vectors and cloning vectors) and hostcells (prokaryotic or eukaryotic) transformed with such vectors.

These vectors can also be used for nucleic acid immunisation [e.g. refs.74 to 85 etc.]. Peptides can be expressed in vivo in this way, as cantherapeutic antibodies. DNA vaccination for the in vivo expression ofpolypeptide mimotopes of carbohydrate antigens is known [e.g. 86].

Host cells which contain nucleic acid of the invention and which expresspolypeptides or antibodies of the invention may be used as deliveryvehicles e.g. commensal bacteria [87]. This is particularly useful fordelivery to mucosal surfaces.

Mimotopes may be useful immunogens in their own right. However, they maybe refined to improve immunogenicity or to improve pharmacologicallyimportant features such as bio-availability, toxicology, metabolism,pharmacokinetics, etc. Mimotopes of the invention can be used fordesigning peptidomimetic molecules [e.g. refs. 88 to 93] withimmunogenic. These will typically be isosteric with respect to themimotopes of the invention but will lack one or more of their peptidebonds. For example, the peptide backbone may be replaced by anon-peptide backbone while retaining important amino acid side chains.

Medical Treatments and Uses

Pharmaceutical compositions of the invention may comprise (a) a glucan(e.g. in the form of a protease-treated cell or a carrier-glucanconjugate), an anti-glucan antibody, a polypeptide comprising a mimotopeof a glucan, a peptidomimetic of the mimotope, and/or a nucleic acidvector encoding the mimotope, and (b) a pharmaceutically acceptablecarrier.

The invention provides a glucan, an anti-glucan antibody, a mimotope ofa glucan, a peptidomimetic of the mimotope, and/or a nucleic acid vectorencoding the mimotope, for use as a medicament.

The invention also provides a method for raising an antibody response ina mammal, comprising administering a pharmaceutical composition of theinvention to the mammal. The antibody response is preferably an IgA orIgG response.

The invention also provides a method for treating a mammal sufferingfrom a microbial infection, comprising administering to the patient apharmaceutical composition of the invention. The infection disease maybe systemic or mucosal.

The invention also provides a method for protecting a mammal against amicrobial infection, comprising administering to the mammal apharmaceutical composition of the invention.

The invention also provides the use of a glucan, an anti-glucanantibody, a mimotope of a glucan, a peptidomimetic of the mimotope,and/or a nucleic acid vector encoding the mimotope, in the manufactureof a medicament for preventing or treating a microbial infection in amammal.

The mammal is preferably a human. The human may be an adult or,preferably, a child. The human may be immunocompromised.

The invention may utilise both (i) an immunogen (e.g. a glucan, a glucanmimotope, a peptidomimetic of the mimotope and/or a nucleic acid vectorencoding the mimotope), and (ii) an anti-glucan antibody or nucleic acidencoding the antibody, in order to provide active and passiveimmunisation at the same time. These may be administered separately orin combination. When administered separately, they will typically beadministered within 7 days of each other. They may be packaged togetherin a kit.

Because glucans (and β-glucans in particular) are an essential andprincipal polysaccharide constituent of almost all pathogenic fungi,particularly those involved in infections in immunocompromised subjects,and also in bacterial pathogens and protozoa, anti-glucan immunity mayhave efficacy against a broad range of pathogens and diseases. Forexample, anti-glucan serum raised after immunisation with S. cerevisiaeis cross-reactive with C. albicans. Broad spectrum immunity isparticularly useful because, for these human infectious fungal agents,chemotherapy is scanty, antifungal drug resistance is emerging and theneed for preventative and therapeutic vaccines is increasinglyrecognized.

The uses and methods of the invention are particularly useful fortreating/protecting against infections of: Candida species, such as C.albicans; Cryptococcus species, such as C. neoformans; Enterococcusspecies, such as E. faecalis; Streptococcus species, such as S.pneumoniae, S. mutans, S. agalactiae and S. pyogenes; Leishmaniaspecies, such as L. major; Acanthamoeba species, such as A. castellani;Aspergillus species, such as A. fumigatus and A. flavus; Pneumocystisspecies, such as P. carinii; Mycobacterium species, such as M.tuberculosis; Pseudomonas species, such as P. aeruginosa; Staphylococcusspecies, such as S. aureus; Salmonella species, such as S. typhimurium;Coccidioides species such as C. immitis; Trichophyton species such as T.verrucosum; Blastomyces species such as B. dermatidis; Histoplasmaspecies such as H. capsulatum; Paracoccidioides species such as P.brasiliensis; Pythiumn species such as P. insidiosum; and Escherichiaspecies, such as E. coli.

The uses and methods are particularly useful for preventing/treatingdiseases including, but not limited to: candidosis, aspergillosis,cryptococcosis, dermatomycoses, sporothrychosis and other subcutaneousmycoses, blastomycosis, histoplasmosis, coccidiomycosis,paracoccidiomycosis, pneumocystosis, thrush, tuberculosis,mycobacteriosis, respiratory infections, scarlet fever, pneumonia,impetigo, rheumatic fever, sepsis, septicaemia, cutaneous and visceralleishmaniasis, corneal acanthamoebiasis, cystic fibrosis, typhoid fever,gastroenteritis and hemolytic-uremic syndrome. Anti-C. albicans activityis particularly useful for treating infections in AIDS patients.

Efficacy of therapeutic treatment can be tested by monitoring microbialinfection after administration of the composition of the invention.Efficacy of prophylactic treatment can be tested by monitoring immuneresponses against β-glucan (e.g. anti-β-glucan antibodies) afteradministration of the composition.

Compositions of the invention will generally be administered directly toa patient. Direct delivery may be accomplished by parenteral injection(e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly,or to the interstitial space of a tissue), or by rectal, oral, vaginal,topical, transdermal, ocular, nasal, aural, or pulmonary administration.Injection or intranasal administration is preferred.

The invention may be used to elicit systemic and/or mucosal immunity.

Dosage treatment can be a single dose schedule or a multiple doseschedule.

The Pharmaceutically Acceptable Carrier

The pharmaceutically acceptable carrier can be any substance that doesnot itself induce the production of antibodies harmful to the patientreceiving the composition, and which can be administered without unduetoxicity. Suitable carriers can be large, slowly-metabolisedmacromolecules such as proteins, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers, andinactive virus particles. Such carriers are well known to those ofordinary skill in the art. Pharmaceutically acceptable carriers caninclude liquids such as water, saline, glycerol and ethanol. Auxiliarysubstances, such as wetting or emulsifying agents, pH bufferingsubstances, and the like, can also be present in such vehicles.Liposomes are suitable carriers. A thorough discussion of pharmaceuticalcarriers is available in ref. 94.

Microbial infections affect various areas of the body and so thecompositions of the invention may be prepared in various forms. Forexample, the compositions may be prepared as injectables, either asliquid solutions or suspensions. Solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection can also beprepared. The composition may be prepared for topical administratione.g. as an ointment, cream or powder. The composition be prepared fororal administration e.g. as a tablet or capsule, or as a syrup(optionally flavoured). The composition may be prepared for pulmonaryadministration e.g. as an inhaler, using a fine powder or a spray. Thecomposition may be prepared as a suppository or pessary. The compositionmay be prepared for nasal, aural or ocular administration e.g. as drops,as a spray, or as a powder [e.g. 95]. The composition may be included ina mouthwash. The composition may be lyophilised.

The pharmaceutical composition is preferably sterile. It is preferablypyrogen-free. It is preferably buffered e.g. at between pH 6 and pH 8,generally around pH 7.

The invention also provides a delivery device containing apharmaceutical composition of the invention. The device may be, forexample, a syringe or an inhaler.

Immunogenic Compositions

Immunogenic compositions comprise an immunologically effective amount ofimmunogen, as well as any other of other specified components, asneeded. By ‘immunologically effective amount’, it is meant that theadministration of that amount to an individual, either in a single doseor as part of a series, is effective for treatment or prevention. Thisamount varies depending upon the health and physical condition of theindividual to be treated, age, the taxonomic group of individual to betreated (e.g. non-human primate, primate, etc.), the capacity of theindividual's immune system to synthesise antibodies, the degree ofprotection desired, the formulation of the vaccine, the treatingdoctor's assessment of the medical situation, and other relevantfactors. It is expected that the amount will fall in a relatively broadrange that can be determined through routine trials. Dosage treatmentmay be a single dose schedule or a multiple dose schedule (e.g.including booster doses). The composition may be administered inconjunction with other immunoregulatory agents.

Even though β-glucans are themselves adjuvants, the immunogeniccomposition may include a further adjuvant. Preferred adjuvants toenhance effectiveness of the composition include, but are not limitedto: (A) aluminium compounds (e.g. aluminium hydroxide, aluminiumphosphate, aluminium hydroxyphosphate, oxyhydroxide, orthophosphate,sulphate, etc. [e.g. see chapters 8 & 9 of ref. 96]), or mixtures ofdifferent aluminium compounds, with the compounds taking any suitableform (e.g. gel, crystalline, amorphous, etc.), and with adsorption beingpreferred; (B) MF59 (5% Squalene, 0.5% Tween 80, and 0.5% Span 85,formulated into submicron particles using a microfluidizer) [see Chapter10 of ref. 96; see also ref. 97]; (C) liposomes [see Chapters 13 and 14of ref. 96]; (D) ISCOMs [see Chapter 23 of ref. 96], which may be devoidof additional detergent [98]; (E) SAF, containing 10% Squalane, 0.4%Tween 80, 5% pluronic-block polymer L121, and thr-MDP, eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion [see Chapter 12 of ref. 96]; (F) Ribi™adjuvant system (RAS), (Ribi Immunochem) containing 2% Squalene, 0.2%Tween 80, and one or more bacterial cell wall components from the groupconsisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM),and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); (G) saponinadjuvants, such as QuilA or QS21 [see Chapter 22 of ref. 96], also knownas Stimulon™; (H) chitosan [e.g. 99]; (I) complete Freund's adjuvant(CFA) and incomplete Freund's adjuvant (IFA); (J) cytokines, such asinterleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.),interferons (e.g. interferon-γ), macrophage colony stimulating factor,tumor necrosis factor, etc. [see Chapters 27 & 28 of ref. 96]; (K)microparticles (i.e. a particle of ˜100 nm to ˜150 μm in diameter, morepreferably ˜200 nm to ˜30 μm in diameter, and most preferably ˜500 nm to˜10 μm in diameter) formed from materials that are biodegradable andnon-toxic (e.g. a poly(α-hydroxy acid) such aspoly(lactide-co-glycolide), a polyhydroxybutyric acid, a polyorthoester,a polyanhydride, a polycaprolactone, etc.); (L) monophosphoryl lipid A(MPL) or 3-O-deacylated MPL (3dMPL) [e.g. chapter 21 of ref. 96]; (M)combinations of 3dMPL with, for example, QS21 and/or oil-in-wateremulsions [100]; (N) oligonucleotides comprising CpG motifs [101] i.e.containing at least one CG dinucleotide, with 5-methylcytosineoptionally being used in place of cytosine; (O) a polyoxyethylene etheror a polyoxyethylene ester [102]; (P) a polyoxyethylene sorbitan estersurfactant in combination with an octoxynol [103] or a polyoxyethylenealkyl ether or ester surfactant in combination with at least oneadditional non-ionic surfactant such as an octoxynol [104]; (O) animmunostimulatory oligonucleotide (e.g. a CpG oligonucleotide) and asaponin [105]; (R) an immunostimulant and a particle of metal salt[106]; (S) a saponin and an oil-in-water emulsion [107]; (T) a saponin(e.g. QS21)+3dMPL+IL-12 (optionally+a sterol) [108]; (U) E. coliheat-labile enterotoxin (“LT”), or detoxified mutants thereof, such asthe K63 or R72 mutants [e.g. Chapter 5 of ref. 109]; (V) cholera toxin(“CT”), or detoxified mutants thereof [e.g. Chapter 5 of ref. 109]; (W)double-stranded RNA; (X) monophosphoryl lipid A mimics, such asaminoalkyl glucosaminide phosphate derivatives e.g. RC-529 [110]; (Y)polyphosphazene (PCPP); or (Z) a bioadhesive [111] such as esterifiedhyaluronic acid microspheres or a mucoadhesive selected from the groupconsisting of cross-linked derivatives of poly(acrylic acid), polyvinylalcohol, polyvinyl pyrollidone, polysaccharides andcarboxymethylcellulose. Other substances that act as immunostimulatingagents to enhance the effectiveness of the composition [e.g. see Chapter7 of ref. 96] may also be used. Aluminium salts (especially aluminiumphosphates and/or hydroxides) are preferred adjuvants for parenteralimmunisation. Mutant toxins are preferred mucosal adjuvants.

Muramyl peptides includeN-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmurarnyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE), etc.

Once formulated, the compositions of the invention can be administereddirectly to the subject. The subjects to be treated can be animals; inparticular, human subjects can be treated. The vaccines are particularlyuseful for vaccinating children and teenagers.

Immunogenic compositions of the invention may be used therapeutically(i.e. to treat an existing infection) or prophylactically (i.e. toprevent future infection). Therapeutic immunisation is particularlyuseful for treating Candida infection in immunocompromised subjects.

As well as β-glucan, the composition may comprise further antigeniccomponents. For instance, the composition may include one or morefurther saccharides. For instance, the composition may comprisesaccharides from serogroups A, C, W135 and/or Y of Neisseriameningitidis. These will typically be conjugated to carrier proteins,and saccharides from different serogroups of N. meningitidis may beconjugated to the same or different carrier proteins. Where a mixturecomprises capsular saccharides from both serogroups A and C, it ispreferred that the ratio (w/w) of MenA saccharide:MenC saccharide isgreater than 1 (e.g. 2:1, 3:1, 4:1, 5:1, 10:1 or higher). Improvedimmunogenicity of the MenA component has been observed when it ispresent in excess (mass/dose) to the MenC component.

The composition may also comprise protein antigens.

Antigens which can be included in the composition of the inventioninclude:

-   -   antigens from Helicobacter pylori such as CagA [113 to 116],        VacA [117, 118], NAP [119, 120, 121], HopX [e.g. 122], HopY        [e.g. 122] and/or urease.    -   a protein antigen from N. meningitidis serogroup B, such as        those in refs. 123 to 129, with protein ‘287’ (see below) and        derivatives (e.g. ‘ΔG287’) being particularly preferred.    -   an outer-membrane vesicle (OMV) preparation from N. meningitidis        serogroup B, such as those disclosed in refs. 130, 131, 132,        133, etc.    -   a saccharide antigen from N. meningitidis serogroup C, such as        the oligosaccharide disclosed in ref. 134 from serogroup C [see        also ref. 135].    -   a saccharide antigen from Streptococcus pneumoniae [e.g. 136,        137, 138].    -   an antigen from hepatitis A virus, such as inactivated virus        [e.g. 139, 140].    -   an antigen from hepatitis B virus, such as the surface and/or        core antigens [e.g. 140, 141].    -   an antigen from hepatitis C virus [e.g. 142].    -   an antigen from Bordetella pertussis, such as pertussis        holotoxin (PT) and filamentous haemagglutinin (FHA) from B.        pertussis, optionally also in combination with pertactin and/or        agglutinogens 2 and 3 [e.g. refs. 143 & 144].    -   a diphtheria antigen, such as a diphtheria toxoid [e.g. chapter        3 of ref. 145] e.g. the CRM₁₉₇ mutant [e.g. 146].    -   a tetanus antigen, such as a tetanus toxoid [e.g. chapter 4 of        ref. 145].    -   a saccharide antigen from Haemophilus influenzae B [e.g. 135].    -   an antigen from N. gonorrhoeae [e.g. 123, 124, 125].    -   an antigen from Chlamydia pneumoniae [e.g. 147, 148, 149, 150,        151, 152, 153].    -   an antigen from Chlamydia trachomatis [e.g. 154].    -   an antigen from Porphyromonas gingivalis [e.g. 155].    -   polio antigen(s) [e.g. 156, 157] such as IPV or OPV.    -   rabies antigen(s) [e.g. 158] such as lyophilised inactivated        virus [e.g. 159, RabAvert™].    -   measles, mumps and/or rubella antigens [e.g. chapters 9, 10 & 11        of ref. 145].    -   antigen(s) from influenza virus [e.g. chapter 19 of ref. 145],        such as the haemagglutinin and/or neuraminidase surface proteins    -   antigen(s) from a paarmyxovirus such as respiratory syncytial        virus (RSV [160, 161]) and/or parainfluenza virus (PIV3 [162]).    -   an antigen from Moraxella catarrhalis [e.g. 163].    -   an antigen from Streptococcus agalactiae (group B streptococcus)        [e.g. 164, 165].    -   an antigen from Streptococcus pyogenes (group A streptococcus)        [e.g. 165, 166, 167].    -   an antigen from Staphylococcus aureus [e.g. 168].    -   an antigen from Bacillus anthracis [e.g. 169, 170, 171].    -   an antigen from a virus in the flaviviridae family (genus        flavivirus), such as from yellow fever virus, Japanese        encephalitis virus, four serotypes of Dengue viruses, tick-borne        encephalitis virus, West Nile virus.    -   a pestivirus antigen, such as from classical porcine fever        virus, bovine viral diarrhoea virus, and/or border disease        virus.    -   a parvovirus antigen e.g. from parvovirus B19.

The composition may comprise one or more of these further antigens.

Toxic protein antigens may be detoxified where necessary (e.g.detoxification of pertussis toxin by chemical and/or genetic means[144]).

Where a diphtheria antigen is included in the composition it ispreferred also to include tetanus antigen and pertussis antigens.Similarly, where a tetanus antigen is included it is preferred also toinclude diphtheria and pertussis antigens. Similarly, where a pertussisantigen is included it is preferred also to include diphtheria andtetanus antigens.

Antigens are preferably adsorbed to an aluminium salt.

Antigens in the composition will typically be present at a concentrationof at least 1 μg/ml each. In general, the concentration of any givenantigen will be sufficient to elicit an immune response against thatantigen.

As an alternative to using proteins antigens in the composition of theinvention, nucleic acid encoding the antigen may be used. Proteincomponents of the compositions of the invention may thus be replaced bynucleic acid (preferably DNA e.g. in the form of a plasmid) that encodesthe protein.

Compositions of the invention may be used in conjunction withanti-fungals, particularly where a patient is already infected. Theanti-fungal offers an immediate therapeutic effect whereas theimmunogenic composition offers a longer-lasting effect. Suitableanti-fungals include, but are not limited to, azoles (e.g. fluconazole,itraconazole), polyenes (e.g. amphotericin B), flucytosine, and squaleneepoxidase inhibitors (e.g. terbinafine) [see also ref. 172]. Theanti-fungal and the immunogenic composition may be administeredseparately or in combination. When administered separately, they willtypically be administered within 7 days of each other. After the firstadministration of an immunogenic composition, the anti-fungal may beadministered more than once.

DEFINITIONS

The term “comprising” means “including” as well as “consisting” e.g. acomposition “comprising” X may consist exclusively of X or may includesomething additional e.g. X+Y.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows immunofluorescence data. The cells are either untreatedfungal ‘Y’ cells (1A, 1C, 1E) or proteinase-treated ‘YDP’ cells (1B, 1D,1F). The labelling antibody is anti-Y-serum 1B), anti-YDP-serum (1C, 1D)or anti-mannoprotein antibody AF 1 (1E, 1F).

FIG. 2 shows mean stimulation index values (±SD) measured experimentalgroups with respect to unstimulated control cultures. Asterisks indicatea significant difference versus the controls (*p<0.05 or **p<0.001, asassessed by ANOVA and Bonferroni's multiple t test). All otherdifferences in proliferative response were not significant.

FIG. 3 shows the results of lethal challenge experiments. FIGS. 3A and3B show surviving animals after challenge. FIG. 3C shows kidneyinfection data.

FIGS. 4 and 5 show the effect of pre-adsorption of sera on passiveimmune transfer.

FIG. 6 is a schematic diagram of a typical C. albicans cell wall showingthe various layers: plasma membrane (PM), zone of mannoprotein (M1),glucan-chitin (GC), glucan (G), mannoprotein (M2) and outer fibrillarlayer (F).

FIG. 7 shows an elution profile of a glucan-CRM197 conjugate, and FIGS.8 to 11 show analysis of the conjugate. FIGS. 8 and 10 are SDS-PAGE gelsof the conjugate (8) before and (10) after ultrafiltration. FIGS. 9 and11 are immunoblots of the conjugate (9) before and (11) afterultrafiltration.

FIG. 12 shows the elution profile of a Bio-Gel P-2 column. Fractionnumbers are shown on the X-axis, with OD_(214nm) being on the Y-axis.

FIG. 13 shows SDS-PAGE analysis of a laminarin-CRM₁₉₇ conjugate.

MODES FOR CARRYING OUT THE INVENTION Preparation ofMannoprotein-Depleted Yeast Cells

C. albicans strain BP, serotype A, from the type collection of theIstituto Superiore di Sanità (Rome, Italy), was routinely maintained onSabouraud agar slants. For all experiments, fungus was cultured in theyeast form in liquid Winge medium at 28° C., washed twice in saline,counted in a haemocytometerer, and resuspended at the desiredconcentration in sterile saline.

For the preparation of normal cells (‘Y cells’) yeast cells suspensions(10⁸ cells/ml) were inactivated at 80° C. for 30 min, washed and storedat 4° C. for no more than a week.

To prepare mannoprotein-depleted cells ('YDP cells'), heat-inactivated Ycells as above (10⁸/ml) were treated with 50 mM DTT in 5 mM EDTANa₂ (1hour, 37° C.). 500 μg/ml Proteinase K (Sigma) was added to the digestionmixture and the cells were treated for one further hour at 37° C. Thefungal cells were extensively washed with saline to remove enzyme,resuspended in saline and used immediately after.

Germ-tube (GT) or hyphal forms of C. albicans were obtained by culturingcells in Lee's medium at 37° C.

Immunisation with Y Cells or YDP Cells

Female, 4 weeks old CD2F1 and SCID mice (Charles River Laboratory,Calco, Italy) were immunised with Y- or YDP-cells. Mice weresubcutaneously injected twice, at weekly intervals, with Y- or YDP-cells(10⁶ cells/100 μl/mouse) in complete Freund's adjuvant (Sigma), and fivetimes by the intraperitoneal route with the same number of immunisingcells without adjuvant. Control animals were injected with Freund'sadjuvant and saline only.

Analysis of the Immune Response

Y-cells contain all of the antigenic cell wall and cytoplasmicconstituents of C. albicans and so they should be able to immunise miceagainst all such antigens. Due to protease treatment, however, YDP-cellsshould not be able to induce a consistent immune reaction againstcell-surface constituents.

To assess antibody responses, immunised animals were bled byretroorbital puncture and sera pooled from each immunisation group wereexamined for antibody content by immunofluorescence assays. Y— orYDP-cells were spotted onto microscope slides and reacted with variousdilution of murine anti-Y or anti-YDP sera or with monoclonal antibodyAF1 (specific for a β-1,2-mannooligosaccharidic epitope which ishighly-expressed on the surface of C. albicans yeast cells) in 0.01MPBS. After extensive washings, slides were treated with FITC-conjugatedanti-mouse IgM antibody and observed with a Leitz Diaplan fluorescencemicroscope.

Anti-YDP serum was strongly reactive in immunofluorescence withYDP-cells (FIG. 1B) but very poorly so with Y-cells (FIG. 1D).Conversely, anti-mannoprotein antibody AF1 reacted with Y-cells (FIG.1E) but not with YDP-cells (FIG. 1F). The surface profile of YDP-cellsis thus very different from that of Y-cells.

Sera were also analysed by ELISA. Polystyrene microtitre plates werecoated with antigens at 50 μg/ml in carbonate buffer, pH 9.6. Plateswere blocked with 3% skim milk in phosphate-buffered saline (PBS),reacted with two-fold dilutions of mouse sera in PBS-0.05%-Tween 20 anddeveloped with alkaline phosphatase-conjugated rabbit anti-mouse IgG orIgM as the secondary antibody and p-nitrophenyl phosphate disodium asthe enzyme substrate. Pooled sera from adjuvant-immunized mice were usedas negative control. Plates were read at 405 nm and antibody titres weredefined as the highest dilution of mouse sera that gave an OD reading atleast twice that of the negative control.

Seven antigens were used:

-   -   C. albicans Y cells (10⁶/well);    -   C. albicans germ-tube cells (10⁶/well);    -   laminarin (β-1,3-glucan, Sigma)    -   pustulan (β-1,6-glucan, CalbioChem);    -   fungal mannoprotein (‘Secr-MP’), prepared from the supernatant        of a 24 hour fungal culture in Lee's medium at 28° C.;    -   mannoprotein fraction MP-F2, purified from the C. albicans cell        wall; and    -   C. albicans soluble glucan antigens (GG-zym), obtained by (i)        preparing glucan ghosts by repeated hot alkali-acid extractions        of fungal cell walls to give purified β-1,3- and glucans        and (ii) digesting the ghosts with β-1,3-glucanase (Zymoliase        100T, Seikagaku) for 1 hour at 37° C.

Results were as follows, with values being from one representativeexperiment out of three performed with similar results:

Antibody titres (×10³) Antigen Anti-Y serum anti-YDP serum Y cells 1.2840 Germ-tubes 1.28 40 β-1,3-glucan 2.56 2.56 β-1,6-glucan 2.56 1.28Secr-MP 2.56 80 MP-F2 >2.56 320 GG-Zym 2.56 2.56

Thus anti-Y-cell serum contained antibodies against all major cell wallconstituents present in both Y and GT forms, including β-1-6 and β-1-3glucans, as well as against major cytoplasmic antigens.

In contrast, and confirming expectations, anti-YDP-cell serum had anelevated titre of anti-glucan antibodies but low antibody titres againstthe whole yeast or germ-tube cells, as well as cell surface-located orsecretory mannoprotein.

To assay the induction of cell-mediated immunity following Y- orYDP-cell immunisation, spleen cells of control or immunised mice wereinduced to proliferate in vitro in the presence of Y- or YDP-cells, aswell as with the β-glucan preparation.

Briefly, splenocyte suspension in 3 ml of 0.16 M Tris-buffered NH₄Cl, pH7.2, were added with 9 ml of complete medium (RPMI 1640, supplementedwith 5% foetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin,1 mM sodium piruvate, 2 mM L-glutamine, MEM-non essential aminoacids,10⁻⁵ M 2-mercaptoethanol). Splenocytes were washed by centrifugation,plated in multiwell plates (10⁶/ml, 200 ml/well) and stimulated with Y-or YDP-cells (10⁵/well), with the GG-zym fraction (50 mg/ml) or withConcanavalin A (2 mg/ml=control). Each condition was assayed intriplicate. Splenocyte cultures were incubated at 37° C. in a 5% CO₂atmosphere. Proliferation was evaluated as ³H-thymidine incorporationafter 4 days for the antigenic stimuli and after 2 days of incubationfor the polyclonal control stimulant. Stimulation indexes werecalculated by dividing mean c.p.m. values of stimulated splenocytecultures with those of unstimulated control cultures.

As shown in FIG. 2, immunisation with Y- or YDP-cells were largelycross-reactive in stimulating a consistent degree of splenocyteproliferation, although a more intense response was seen withsplenocytes stimulated in vitro with the specific immunising antigenicpreparation. The splenocytes of all animals, including the non-immunisedcontrols, responded to the polyclonal stimulation with ConA. Noproliferation was detected in splenocyte cultures stimulated in vitrowith β-glucan of C. albicans.

Overall, therefore, immunisation with Y- or YDP-cells induced largelycross-reactive humoral and CMI responses to antigens present on bothcellular preparations. However, anti-MP and anti-Y-cell surface directedantibodies were present only in mice immunised with whole Y cells.

Protection Against Lethal Challenge

Having demonstrated that immunisation with Y cells induced consistenthumoral and cell-mediated immune responses against major antigenicconstituents of the fungus, the protective capacity of cells was testedin an acutely lethal mouse candidiasis model.

Protection was evaluated by monitoring animal survival (15 per group)for 60 days after intravenous challenge with a lethal dose of C.albicans. The dose was either 1×10⁶ (FIG. 3A) or 2×10⁶ (FIG. 3B) cell in0.1 ml, or an adjuvant-only control.

Mice in the non-immunised control group had a median survival time of1-2 days at the higher dose (FIG. 3B). Mice immunised with Y-cellsshowed an increased median survival time to the fungal challenge but hadall died by day 15-17 after challenge and overall survival rates werenot statistically different from the controls.

In contrast, animals immunised with YDP cells were much more resistant,with median survival >60 days. Differences in survival rates ofYDP-immunized animals compared to adjuvant-treated animals and to Y-cellimmunised animals were statistically significant (p<0.05, Fisher exacttest) at both doses.

Protection was also evaluated by quantifying the extent of Candidaoutgrowth in the kidneys of animals infected with 10⁶ cells. Thisinvolved aseptic removal of the left kidney of sacrificed mice at day 7post-challenge followed by homogenisation in sterile saline containing0.1% Triton-X100 (Sigma). The number of colony forming units (CFU) perorgan was determined by a plate dilution method on Sabouraud dextroseagar. Each kidney was examined separately and at least three distinctdilution from each sample were assayed in triplicate.

As shown in FIG. 3C, mean fungus burden in the kidney was much lower inthe YDP-cell immunised mice (CFU <10³) than in the Y-cell immunisedgroup (15.4±0.6×10³; p<0.05 by Kruskal-Wallis ANOVA and Bonferroni-typenon parametric multiple comparison) and in the control group (˜60×10³;p<0.05). The difference between the Y-cell and control groups was notstatistically significant.

Experiments were also performed with SCID mice with the same schedule ofvaccination as for immunocompetent animals. No protection was observed,demonstrating that adaptive immune responses are essential forprotection. Unlike the reports in reference 173 for C. neoformans,therefore, CD4⁺ cells are not involved in the antibody-mediatedprotection.

Passive Immunisation

As a major difference in the immune response to Y- or YDP-cells was inthe antibody specificities to cell wall constituents, the ability ofimmune sera to transfer protection to non-immune animals was tested.These experiments also evaluated the potential contribution of theimmune system of the recipient mice to the protection conferred by thepassively-administered serum.

CD2F1 or SCID mice were passively immunised by a single intraperitonealinjection of 0.5 ml of anti-Y— or anti-YDP-cell serum. Control animalsreceived serum from adjuvant-immunised mice. Each serum was heat-treated(56° C., 30 min) before transfer to inactivate heat-labile, non-antibodyconstituents. Mice were intravenously challenged two hours aftertransfer of sera with a sublethal dose of C. albicans (5×10⁵ cells) andprotection was evaluated two days later using the kidney model asdescribed above. These experiments were performed by using variousbatches of serum from animals independently immunised with the YDP- orY-cell vaccine.

Results at 2 days post-challenge were as follows, with data representingweighted means of individual CFU counts enumerated from each group ofmice. Statistical analysis was by Kruskal-Wallis ANOVA followed bynon-parametric Bonferroni-type multiple comparison test:

Recipient Kidney fungal burden mouse Pre-challenge treatment (CFU × 10⁻³± SD) p CD2F1 Control (Adjuvant only) serum 361.7 ± 17.6    —anti-Y-cell serum 1 393.8 ± 7.7 n.s. {close oversize brace} <0.01anti-YDP-cell serum 1 8.7 ± 0.4 <0.05  SCID Control (Adjuvant only)serum 214.4 ± 1.8    — anti-Y-cell serum 2 392.2 ± 7.7 n.s. {closeoversize brace} <0.05 anti-YDP-cell serum 2 44.2 ± 0.8 0.05 n.s. = notsignificant

Thus animals receiving anti-Y-cell serum had the same elevated fungusburden in their kidney as those receiving the control non-immune serum.In contrast, those receiving the anti-YDP cell serum had significantlyfewer fungal cells in their kidney than the animals receiving controlserum. This was observed with different batches of respective immunesera, and in both the immunocompetent and the SCID mice.

As these data suggested that antibodies play a significant role inprotection, the IgM fraction of serum from the YDP-immunised mice waspurified and used for passive immunisation. The same fraction purifiedfrom the serum of animals given adjuvant only was used as a control. Ina single experiment, the fungus kidney burden on day 2 post-challenge offour mice intravenously injected 10⁶ cells of C. albicans was 290±8(×10³) cells against 1359±18 (×10³) cells in the kidney of control mice(p<0.01). The IgM fraction of YDP serum was highly reactive againstglucan extract of C. albicans.

Removal of Passive Immunisation Efficacy

Serum antibodies generated by immunisation with YDP-cells recognizeβ-glucan constituents (see above). These antibodies were removed andpassive transfer of immunity was re-tested.

Anti-Y or anti-YDP-cell sera were selectively adsorbed to removeglucan-specific or anti-surface mannoproteins antibodies. Sera (2 ml)were treated (1 hour, 0° C.) with 10 mg of particulate glucan (glucanghosts) or with 2×10⁸ live yeast cells of C. albicans. Adsorbants wereremoved by centrifugation, and the procedure was repeated three times.Efficacy of the adsorption procedure was evaluated by ELISA, using yeastcells or GG-zym as the coating antigens.

This procedure typically lowered the anti-β-glucan titres of anti-YDPsera and the anti-MP titre of anti-Y sera by 2 to 3 logs. Antibodiesagainst β-glucan were not removed by adsorption with intact Y cells.

The effect of pre-adsorption on YDP-sera was also assessed in the kidneyburden model (FIG. 4). Unadsorbed or pre-adsorbed sera (0.5 ml/mouse)were given i.p. to mice (three per group) two hours before anintravenous sublethal challenge with C. albicans (5×10⁵ cells/mouse).Kidney invasion was assessed 48 hours post-challenge by individual CFUcounts.

YDP-serum was much better (p<0.05) than the control serum, butpre-adsorption with β-glucans removed this effect (p<0.05), with nostatistically significant different between control serum andadsorbed-serum.

Therefore an appreciable level of protection can be transferred to naiveanimals by the serum of YDP-cell recipient animals, the protective serumfactor is heat-stable, and the immunoglobulin fraction of the serum isalso protective. The protective serum is rich in anti-β-glucanantibodies and poor in anti-MP antibodies. When adsorbed on pureβ-glucan, the serum loses much of its protective capacity. Moreover, theanti-Y-cell serum was protective when the anti-mannoprotein but not theanti-β-glucan antibodies were lost. Overall, this evidence suggests thatprotective IgM include those recognizing β-glucan.

Protective and Non-Protective Antagonistic Antibodies

The previous data suggest that anti-β-glucan antibodies play a role inthe protection conferred by the YDP-cell vaccine. However, the sera ofanimals immunised with the Y-vaccine also contain high titres ofanti-β-glucan antibodies (see above). Thus the Y-sera may contain asubstance, not present in the YDP-sera, which inhibits the activity ofthe anti-β-glucan antibodies.

Given the differences between Y-cells and YDP-cells, and between theirsera, the substance appeared to be antibody against fungal cell surfaceconstituents. To test this hypothesis, sera from Y-cell-immunisedanimals were adsorbed to Y-cells and the resulting sera wereadministered to SCID mice. The Y-cell-adsorbed sera had a substantialreduction of anti-MP antibodies but maintained elevated anti-β-glucanantibody levels. As shown in FIG. 5, animals receiving pre-adsorbed Ysera (column 5) had a kidney burden of about 2 logs lower than animalsgiven non-adsorbed Y sera (column 4), and this was comparable to that ofanimals given the protective YDP-sera (column 2).

Thus the Y-serum contains antibodies to the yeast cell surface which areinhibitory for protection conferred by antibodies against underlyingcell wall antigens (β-glucan).

These data may explain why anti-Candida sera have been found to beinconsistent in transferring protection, and why immunisation with wholeinactivated cells of C. albicans has been variably protective thoughalways stimulating an elevated DTH, cell-mediated immunity and abundantanti-Candida antibodies. The data strongly suggest thatantibody-mediated protection against C. albicans not only requires thepresence of the right antibody but also requires the absence of certainother antibodies.

As antibodies against abundantly-expressed cell-surface constituents areprevalent in healthy people colonized by C. albicans, the generation ofantagonistic or blocking antibodies may be a mechanism by which thefungus defends itself from the eradicating capacity of other antibodies.

Antibodies to β-glucan have previously been observed in normal humansera [e.g. 174]. As they do not react with cell surface components,however, and do not obviously opsonise fungal cells, a role in themechanism of protection had been dismissed. The anti-β-glucan IgG2 ofreference 174 were specifically said to be dispensable for opsonicactivity of non-encapsulated, β-glucan-exposing C. neoformans cells. Thedata herein invite a reconsideration of this view, as anti-β-glucanantibodies are shown to play a role in protection, at least whenblocking antibodies are absent.

Even if blocking antibodies are present, the levels of anti-mannoproteinantibodies are higher than the levels of anti-glucan antibodies duringnatural infection and colonisation, but administering immunogenicglucans may tip the balance of inhibitory and protective antibodies infavour of protection. Furthermore, anti-C. albicans blocking antibodiesmay not inhibit the activity of anti-glucan antibodies against otherpathogens (e.g. those whose cell walls contain glucan but notmannoprotein).

Preparation of Glucan-Carrier Conjugates

As described above, GG-zym is prepared by glucanase digestion of aglucan ghost preparation of C. albicans cells. GG-zym is pure (>99%)β-glucan. GG-zym saccharide was conjugated to CRM197 carrier protein togive ‘CRM-GG’.

The conjugation process used to prepare CRM-GG starts with a reductiveamination reaction by which one terminal amino group is added per chain.This amino group is subjected to reaction with di-N-hydroxysuccinimideester of adipic acid to give an activated linker. The activatedsaccharides are conjugated to CRM197 protein and the conjugateintermediate is purified by ultrafiltration.

Reductive amination was performed by reacting an aqueous β-glucansaccharide solution (2 mg/ml GG-zym) with ammonium acetate (300 g/l) inthe presence of sodium cyanoborohydride (28.9 g/l). The acetate andcyanoborohydride were added to the saccharide solution by funnel and themixture was stirred until the components dissolved. pH was then adjustedto 7.2 and the mixture was transferred into a glass bottle which wassealed and incubated in a 50±1° C. water bath for 5 days. This reactiongave saccharide with a terminal amino group.

The aminated saccharide was then purified by chromatography on aSephadex G-10 column. All chromatography was performed at roomtemperature using a flow rate of 24 cm/hr, and progress was monitored byconductivity and by absorbance at 214 nm. The column was initiallywashed with 2 litres (5 column volumes) distilled water in order toremove the 20% ethanol storage solution. The column was thenequilibrated with 2 litres of 0.2M NaCl. Sample was loaded onto thecolumn and fractions were collected (FIG. 7). As the saccharide has noabsorbance at 214 nm, fractions were analysed by glucose analysis(phenol sulphuric method [175]) and fractions containing the saccharidewere combined. The saccharide eluted from the column after 1.5 columnvolumes of 0.2M NaCl.

The purified product was concentrated and purified to remove NaCl.Membranes (1K microsep, PALLFILTRON) were washed with distilled water bycentrifuging at 3000 rpm for 1 hour at 4° C. on a minifuge T. Saccharidewas added to the membranes and centrifuged for 3 hours at 4000 rpm togive a 0.5 ml volume. 1.5 ml distilled water was added and the mixturewas centrifuged as before. This cycle was repeated until the NaClconcentration was lower than 0.02M. Samples were collated. In addition,the membranes were given a final wash with distilled water and the washsolution was added to the collated samples. The purified saccharide wasanalysed for glucose [175] and for amine groups [176].

The saccharide was then dried by rotary evaporation using a Buchirotoevaporator (Model EL 131; 90 rpm) in conjunction with a KNFNeuberger Laboport vacuum pump, a Buchi 461 water bath (37° C.) and aPharmacia Biotech multitemp III recirculating condenser chiller (4° C.).Vacuum pressure was increased slowly in order to avoid boiling. In afirst phase of evaporation liquid was visible. Near the end of thisphase, the majority of product appeared dry, with some bubbles withinwhich liquid could be seen moving. The first phase ended when no obviousliquid was seen moving. The second phase of drying was an additionaltime under the same conditions until the material looked glassy andcracked

The dried saccharide was then activated by reacting its free amino groupwith the di-N-hydroxysuccinimide ester (bis-NHS ester) of adipic acid.The saccharide was dissolved in DMSO to give an amine concentration of40 mmol/L. Triethylamine (TEA) was added at a TEA:amine volume ratio of1.113 and the mixture was stirred to homogeneity.

Succinic acid diester was dissolved in DMSO, using five times the volumeof DMSO which was used to dissolve the saccharide. The amount ofsuccinic acid diester was calculated to give a 12:1 molar ratio ofsuccinic diester:amine groups.

With the succinic acid diester solution stirring, the saccharide mixturewas slowly added and then incubated at room temperature with stirringfor 1.5 to 2 hours, after which the reaction mixture was slowly added toroom temparature dioxane (4 volumes in polypropylene centrifuge bottles)with stirring in order to precipitate the activated saccharide andseparate it from DMSO, bis-NHS ester and TEA. After 75 minutes forprecipitation, the bottles were capped and stirred for 10 minutes. Themixture was then centrifuged at 7000 g for 20 minutes at 15° C. Thesupernatant was decanted and the dioxane washing was repeated for atotal of 5 washes. The mixture was then dried using a vacuum dryer(Lyovac GT 2). The dried saccharide was analysed for active ester [177].

For conjugation, activated saccharide ester and CRM197 were mixed at aproportion of 20 mmol activated saccharide per mmol CRM197 in 0.01Msodium phosphate buffer, pH 7,2. The protein solution was adjusted togive a CRM197 concentration of 45 g/l and was stirred slowly in a glassbottle with a magnetic stir bar. Activated saccharide was slowly addedto the bottle, which was then capped. The stirring rate was adjustedsuch that a small vortex formed without excess foaming. Conjugationproceeded for 14 to 22 hours. The final product was analysed by SDS-PAGE(FIG. 8; 1: MW markers; 2: CRM197; 3: conjugate; 4: supernatantconjugate) and by western blot using anti-CRM antibodies (FIG. 9; 1:supernatant conjugate; 2: conjugate; 3: CRM197).

Finally, the conjugate was purified for immunogenicity studies usingultrafiltration membranes with a nominal 100 KDa cut-off (Membranes 100KMicrocon SK, Amicon). Membranes were washed with 0.5 ml distilled waterby centrifuging (Biofuge Picot) for 10 minutes at 2500 rpm: Theconjugate was then added and centrifuged for 3 minutes at 13000 rpm. Thesupernatant was removed, re-added to the membrane and centrifuged for 25minutes at 2500 rpm. 0.3 ml 0.01M sodium phosphate buffer (pH 7.2) wasadded and centrifuged for 25 minutes at 2500 rpm. This was repeated fora total of 7 times. The final purified product was analysed for protein[178], for glucose (high pressure anion exchange chromatography withpulsed amperometric detection), by SDS-PAGE (FIG. 10; 1: CRM197; 2:purified conjugate), and by western blot using anti-glucan antibodies(FIG. 11).

Analysis of GG-zym

The GG-zym β-glucan preparation was investigated by gel filtrationchromatography and by ¹H & ¹³C NMR. It was found to contain two β-glucanfractions, each representing around 50% of the GG-zym antigen weight:

-   -   Pool 1 contains basically β-1,6-glucan chains with ramifications        of β-1,3-chains. The approximate degree of polymerisation (DP)        of the β-1,6 chains is 36 glucose monosaccharide units, while        that of the β-1,3 chains is approximately 9-10 monosaccharide        units. The degree of branching (DB) is approximately 0.6.    -   Pool 2 contains short β-1,3-glucan chains with few        β-1,6-linkages. DP is approximately 3.9 with a DB of        approximately 0.03.

Immunogenicity of Conjugates

CRM-GG was tested by ELISA against immune sera from mice immunised withYDP-cells. The conjugate was highly reactive with all assayed sera,demonstrating antigenic equivalence of CRM-GG to the glucan expressed onC. albicans cells.

To test immunogenicity of the conjugate it was administered to CD2F1mice according to three schedules:

-   Schedule A) 7 mice were each inoculated intraperitoneally with    CRM-GG conjugate (20 μg protein). After 21 days, a pool of sera    obtained from all immunised animals was tested by indirect ELISA. No    mouse showed sign of suffering or illness during immunisation.-   Schedule B) 7 mice were inoculated subcutaneously on day 0 and day 7    with CRM-GG conjugate (10 μg as protein) in incomplete Freund's    adjuvant. An intraperitoneal boost was given on day 28 using 10 μg    conjugate without adjuvant. Serum were pooled 7 days later and    tested as for schedule A. During schedule B some animals were found    suffering and one died.-   Schedule C) 12 mice were inoculated subcutaneously on day 0 with    CRM-GG conjugate (10 μg as protein) in complete Freund's adjuvant.    Intraperitoneal boost was given on day 28 using 10 μg conjugate    without adjuvant. Sera were collected 3 days later and pooled for    analysis as above. No mouse showed sign of suffering or illness    during immunisation.

As a negative control, un-conjugated CRM197 was administered accordingto Schedule B. Serum raised against unconjugated GG-zym in mouse usingmultiple aggressive immunisations was used as a positive control foreliciting antibody responses (2×10 μg intranasal instillation with 1 μgcholera toxin adjuvant, followed by five weekly i.p. infections of 50 μgantigen).

IgM and IgG titres of sera from immunised animals were determined byindirect ELISA using specific alkaline phosphatase-conjugated anti-mouseIgM or anti-mouse IgG secondary antibodies. Results (OD readings) forschedules A & B were as follows:

Coating antigen GG-ZYM CRM SERUM DILUTION anti-IgM anti-IgG anti-IgMAnti-IgG anti-GG- 1:30 1.15 — 0.13 0.39 CRM 1:60 0.91 — 0.10 0.30(schedule A) 1:120 0.69 — 0.09 0.22 1:240 0.43 — — 0.17 1:480 0.28 — —0.12 1:920 0.18 — — 0.10 1:1920 0.14 — — 0.10 anti-GG- 1:30 1.28 — 0.940.84 CRM 1:60 1.22 — 0.69 0.83 (schedule B) 1:120 1.18 — 0.47 0.77 1:2401.1 — 0.31 0.76 1:480 0.93 — 0.21 0.73 1:960 0.68 — 0.13 0.68 1:1920 0.5— 0.10 0.53 anti-CRM 1:30 0.44 0.09 0.72 0.93 (negative 1:60 0.28 — 0.780.8  control) 1:120 0.21 — 0.64 0.65 1.240 0.16 — 0.50 0.48 1.480 0.13 —0.31 0.30 1:960 0.11 — 0.21 0.20 1:1920 0.1 — 0.20 0.15 anti-GG-zym 1:300.94 — 0.09 0.09 (positive 1:60 0.75 — ″ ″ control) 1:120 0.54 — ″ ″1:240 0.51 — ″ ″ 1:480 0.33 — ″ ″ 1:960 0.22 — ″ ″ 1:1920 0.16 — ″ ″

For schedule C, antibody ELISA titres (the highest dilution givingOD_(405 nm) value at least 2× control) and isotones were as follows:

Coating antigen (50 μg/ml) GG-ZYM Pool 1 Pool 2 Pustulan Laminarin SerumIgM IgG IgM IgG IgM IgG IgM IgG IgM IgG Anti-GG-CRM >1920 >1920 >19201920 1920 480 480 >>1920 960 <15 Anti-GG-zym 240 <15 480 15 120 <15 120<15 120 <15

Appreciable anti-CRM antibody titres, particularly IgG, were thusobtained following immunisation with the conjugate. More importantly,immunization also induced elevated anti-GG-zym antibody titres. Usingschedules A and B, antibodies were exclusively of the IgM isotype. Usingschedule C, however, animals showed consistent production of IgGantibodies against GG, in particular against the β-1-6 glucans ofpool 1. Thus conjugation transformed a poor immunogen into a strong one,conferring isotype switching, and memory responses.

Analysis of Immune Responses

Sera obtained against the GG-CRM conjugate (schedule B) were testedagainst each pool to see if either was dominant. The same positivecontrols were used as before.

Indirect ELISA results were as follows, with values indicatinganti-GG-CRM serum titres (IgM):

Sera Pool 1 Pool 2 anti-GG-CRM >2500 160 anti-GG-zym 320 40

ELISA inhibition results were as follows:

SERUM INHIBITOR Dose (μg/ml) % inhibition anti-GG-CRM Pool 1 60 93(1:250 dilution) 30 92 15 90 0 0 Pool 2 60 62 30 53 15 30 0 0anti-GG-zym Pool 1 60 82 (1:80 dilution) 30 82 15 80 0 0 Pool 2 60 63 3055 15 42 0 0

Thus the conjugate CRM-GG mainly induces antibodies against β-glucanchains present in Pool 1 i.e. higher molecular weight, primarilyβ-1,6-glucan.

Overall, the data obtained by mouse immunisation with the CRM-GGconjugate show that the conjugate is highly immunogenic and that theantibody response is largely superior, in terms of antibody titres, tothat obtained with the GG-Zym polysaccharide alone. Importantly,antibody obtained after CRM-GG conjugate immunization has the sameantigenic specificity as protective anti-β-glucan antibodies.

Cross-Reactive Immune Responses

GG-zym is derived from C. albicans. Mice were immunised with YDP cellsof either C. albicans or S. cerevisiae using the same schedule asdescribed above for YDP cells and the resulting sera were tested byELISA for reactivity to GG-zym. Titres are the highest serum dilutionwith a reading twice that of the well without coating antigen. Thesecondary antibody was rabbit anti-mouse IgM.

Coating antigen Serum GG-Zym Laminarin Pustulan Adjuvant only <20 <20<20 YDP-C. albicans >2560 320 >2560 YDP-S. cerevisiae >2560 >2560 >2560

Antibodies raised against S. cerevisiae YDP cells are thuscross-reactive with C. albicans GG-zym antigen. The immune responsesagainst S. cerevisiae and C. albicans are not identical, however, asanti-C. albicans serum is much less reactive with laminarin than theanti-S. cerevisiae serum.

Alternative Conjugation Process

The glucan purification and conjugation process described above wasrepeated with one or both of the following changes:

-   -   Rather than use 0.2M NaCl for oligosaccharide purification after        reductive amination, 20 mM NaCl was used. The alternative        process involves reduced salt concentration after gel filtration        and improves downstream oligosaccharide activation. The aminated        saccharide elutes from the Sephadex G-10 column after 1.5 column        volumes of 20 mM NaCl.    -   For conjugate purification, the first conjugate was purified (as        before) by ultrafiltration using membranes with nominal 100 kDa        cut-off. Other conjugates were purified by ultrafiltration using        membranes with either a nominal 30 kDa or nominal 50 kDa        cut-off, depending on the characteristics of the conjugates—for        cross-linked high MW conjugates the 50 kDa membrane was used;        for conjugates without cross-linking the 30 kDa membrane was        used. The 30 kDa & 50 kDa membranes were used with either a        Centricon™ (centrifugal filter unit) technique or a tangential        flow technique.    -   For the Centricon™ technique, 30 kDa or 50 kDa membranes were        obtained from Millipore™ for use with a Minifuge T (Heraeus        Sepatech). The device was washed by centrifuging 3 ml distilled        water for 10 minutes at 3500 rpm. The conjugate was then        centrifuged at 3500 rpm for 3 minutes. The supernatant was        removed and added to the device followed by 25 minutes of        centrifugation at 3500 rpm. 1.5 ml 0.01M sodium phosphate buffer        (pH 7.2) was then added, and centrifuged for 25 minutes at 3500        rpm. This procedure was performed 8 times in total.    -   For tangential flow ultrafiltration, a Holder Labscale        (Millipore) apparatus was used with 505U Pumps (W. Marlow) and        PLCIC-C 30 kDa cut-off 50 cm² membranes (Millipore). The system        was washed with distilled water until the pH of the permeate was        <7.00. The system was then equilibrated with 100 ml of 0.01M        sodium phosphate buffer (pH 7.2). The sample was then loaded        into the holder and the following ultrafiltration conditions        were applied: pressure in 25.7 psi {1 psi=6894.757 Pa}; pressure        out 18.4 psi {1 psi=6894.757 Pa}; flow rate 7.6 ml/min. Forty        diafiltration volumes of 0.01M sodium phosphate buffer (pH 7.2)        were used. Finally, the sample was placed in another vessel and        the system was washed, first with 0.1 M NaOH, then with water        and finally with 0.05M NaOH.

Separation of Pools 1 & 2

As mentioned above, the GG-zym β-glucan preparation contains two mainfractions—pools 1 & 2. These two pools can be separated by gelfiltration.

Using a Pharmacia™ FPLC system operating at room temperature and a flowrate of 0.37 cm/hr, a Bio-Gel P-2 Fine column (Bio Rad) was equilibratedwith 450 ml of 0.02M PBS (pH 7.4). The mixed GG-zym sample was loadedonto the column and eluted with 1.0 column volume of 0.02M PBS (pH 7.4).After collecting fractions, the column was stripped with the same bufferfor 1.5 column volumes, then washed with 3 column volumes of distilledwater and with 3 column volumes of 20% ethanol as storage solution. Theoutput from the column was monitored by conductivity and by absorbanceat 214 nm, as described above. As shown in FIG. 12, pools 1 and 2 eluteseparately.

The two different glucan populations can be used separately to makeconjugates using the procedure described above.

Laminarin Conjugate

For comparative purposes, a further conjugate was made using CRM197carrier and laminarin. Laminarin has a similar glucan structure to pool2 of GG-zym (i.e. 1,3-β-glucans), but it has a higher average degree ofpolymerisation of about 30.

The for making the laminarin conjugate is the same as used for theGG-Zym, except for oligosaccharide purification after reductiveamination. For laminarin, the process used a Holder Labscale apparatus(Millipore) using 505U Pumps (W. Marlow) and a PLCBC-C 3 KD cut-off 50cm² membrane (Millipore). The apparatus was washed with distilled wateruntil the pH of the permeate is <7.00. The apparatus was thenequilibrated with 100 ml of 0.5M NaCl, and the sample was then loadedinto the holder and the following ultrafiltration conditions wereapplied: pressure in 19 psi {1 psi=6894.757 Pa}; pressure out 13 psi {1psi=6894.757 Pa}; flow rate 0.6 ml/min. Thirteen diafiltration volumesof 0.5M NaCl were used, followed by six diafiltration volumes of H₂O.Finally, the sample was placed in another vessel and the system waswashed, first with 0.1 M NaOH, then with water and finally with 0.05 MNaOH.

FIG. 13 shows SDS-PAGE of the laminarin conjugate. The two spots at theleft show unconjugated CRM¹⁹⁷ carrier, and the two spots at the rightshow the conjugate.

SUMMARY

Whole intact C. albicans yeast cells do not confer protective immunityagainst C. albicans, whereas protease-treated cells can conferprotective immunity.

Anti-Candida protection induced by protease-treated cells is in partmediated by antibodies, with anti-β-glucan antibodies playing animportant role.

Protective, serum-transferable, antibody-mediated protection against C.albicans can be negated by immune responses to cell-surface located,immunodominant fungal antigens. Thus immunisation with whole intactcells elicits cell-surface reactive, antagonistic or blockingantibodies.

Protein-glucan conjugates are effective immunogens.

Anti-glucan antibodies raised against one organism can cross-react withglucans from another organism.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

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1. An immunogenic composition comprising an immunogenic componentconjugated to a pharmaceutically acceptable protein carrier, wherein (a)the immunogenic component is a glucan polymer that has a molecularweight of less than 100 kDa, wherein said glucan is i) a glucan from aprotease-treated fungal cell, ii) a glucan from a mannoprotein depletedfungal cell, or iii) a pure glucan; and (b) when the composition isadministered to a mammal, it elicits protective anti-glucan antibodiesbut does not elicit antibodies which inhibit the protective efficacy ofthe anti-glucan antibodies.
 2. The composition of claim 1, wherein theglucan is a β-glucan.
 3. The composition of claim 2, wherein the glucancontains one or more β-1,6-linkages.
 4. The composition of claim 1,wherein the glucan is a fungal glucan.
 5. The composition of claim 1,wherein the glucan is on the surface of a protease-treated fungal cell.6. The composition of claim 2, wherein the glucan is a β-glucan derivedfrom the cell wall of a Candida.
 7. The composition of 6, wherein theglucan is a β-glucan derived from the cell wall of C. albicans.
 8. Thecomposition of claim 1, wherein the composition is substantially free ofmannoprotein.
 9. The composition of claim 1, further comprising anadjuvant.
 10. The composition of claim 1, wherein the composition isdepleted of mannoprotein.
 11. The composition of claim 10, wherein thecomposition is depleted of mannoprotein by treatment of a fungal cellwall with a protease.
 12. An immunogenic composition comprising animmunogenic component conjugated to a pharmaceutically acceptableprotein carrier, wherein the immunogenic component is a glucan on thecell wall of a fungal cell, wherein said cell wall is depleted ofmannoprotein.
 13. An immunogenic composition comprising an immunogeniccomponent conjugated to a pharmaceutically acceptable protein carrier,wherein the immunogenic component is a glucan on the cell wall of aprotease-treated fungal cell.
 14. A method for raising an antibodyresponse in a mammal, comprising administering a composition accordingto claim 1 to the mammal.
 15. A method for treating or preventing amicrobial infection in a mammal, comprising administering to the mammala composition according to claim
 1. 16. The method claim 14, wherein themammal is a human.
 17. The method of claim 15, wherein the mammal is ahuman.
 18. The method of claim 15, wherein the microbial infection iscaused by Candida.
 19. The method of claim 18, wherein the microbialinfection is candidosis.